1. Field of the Invention
The present invention relates to a method of constructing a DNA library having increased proportion of a desired nucleic acid(s) therein by removing, from a parent DNA library, a nucleic acid(s) other than the desired nucleic acid(s) using RecA protein.
2. Description of the Related Art
A DNA library, particularly a cDNA library, is a very useful tool for gene cloning. To date, various genes have been cloned from cDNA libraries. A cloned gene is used not only to determine its nucleotide sequence but also to determine amino acid sequence of a protein encoded by the gene and to produce a large amount of the protein in bacterial or yeast cells.
However, cDNAs that can be easily cloned from a cDNA library are restricted to those whose template mRNAs are abundantly expressed in a cell. Hence, cDNAs easy to be cloned have now mostly been cloned, and it is getting harder to clone a novel cDNA with high efficiency.
In order to clone a novel cDNA efficiently from a cDNA library, it is necessary to remove cDNAs already cloned from the library. For this purpose, the following prior arts have been devised.
Subtractive hybridization has been used primarily for this purpose. In the method, mRNAs are harvested both from cells (or tissues) expressing a gene(s) of interest and cells not expressing. Then, from mRNAs of the former cells, cDNAs are synthesized. Through hybridization of the cDNAs with the mRNAs from the latter cells, cDNAs that are present in both cells are selectively removed. This enables enrichment and isolation of a gene(s) specifically expressed in a tissue or a cell.
xe2x80x9cGenome Res. 1996 Sep: 6(9): p.791-806xe2x80x9d discloses subtractive hybridization using a hydroxyapatite column. In this method, primers from vector-derived sequences are elongated using a single-stranded DNA library as templates. After denaturation and annealing, DNAs that form a double strand again are specifically removed by a hydroxyapatite column. Since probability of annealing is dependent on concentration, abundant clones are preferentially removed.
However, this method can only be applied to a relatively short cDNA of approximately 0.4-2.5 kb. Because non-specific hybridization is likely to occur, when a cDNA library is used which includes a long sequence having an insert size greater than 3 kb. A long sequence is often found to be a functionally important gene encoding a multifunctional protein or a protein with complex conformation. Therefore, it is a major drawback of this method that it cannot be applied to a library containing a long sequence. Furthermore, this method cannot distinguish even short cDNAs, if sequences of the cDNAs are derived from an identical gene and having common sequences at 3xe2x80x2 and 5xe2x80x2 ends but not in their central regions.
Other method widely used for a similar purpose is differential hybridization.
In this method, cDNA probes are synthesized with mRNAs prepared from control cells and cells of interest from which a specific gene is obtained. A cDNA library generated from the cells of interest is then plated, and colonies on one plate are replica-plated onto two filters. For one filter, hybridization is performed with cDNA probes from the cells of interest. For the other, hybridization is performed with cDNA probes from the control cells. cDNAs specific for the cells of interest can be detected by comparing the results.
However, in this method, differences in hybridization between two filters must be compared from colony to colony. Accordingly, it is difficult to deal with numerous colonies by means of the method. This method is thus not suitable for reconstruction of a whole library. This method has also a drawback of being time-consuming for checking many possible pseudo-positive or pseudo-negative signals.
To overcome such drawbacks of this method, xe2x80x9cMethods in Enzymology 1995: 254: p.304-321xe2x80x9d discloses differential display method, which is a combination of conventional differential hybridization and polymerase chain reaction (hereinafter referred to as xe2x80x9cPCRxe2x80x9d). However, this method can detect a difference in a pattern only when a difference in an expression level is significant. Furthermore, since this method cannot produce clones directly, it is necessary to select clones by any method based on a PCR product.
The present invention is made to solve above problems resided in prior arts. Accordingly, the object of the invention is to provide a method that is capable of specifically enriching a desired DNA with a long insert size in a DNA library, and directly providing a clone of the DNA.
To solve the above problems, the invention provides a method of constructing a DNA library having increased proportion of a first double-stranded DNA to be raised in its ratio by removing, from a parent DNA library, a second double-stranded DNA that is not identical to the first double-stranded DNA, the method comprises the following steps of;
(a) converting the first double-stranded DNA and the second double-stranded DNA in the parent DNA library into a corresponding first single-stranded DNA and a second single-stranded DNA respectively, to prepare a library containing the first single-stranded DNA and the second single-stranded DNA;
(b) adding, to the library prepared in the step (a), RecA protein and a linear double-stranded DNA homologous to the second single-stranded DNA to regenerate the second double-stranded DNA from the second single-stranded DNA thereby preparing a library containing the first single-stranded DNA and a regenerated second double-stranded DNA;
(c) removing the regenerated second double-stranded DNA from the library prepared in the step (b);
(d) regenerating the first double-stranded DNA from the first single-stranded DNA thereby constructing a DNA library having increased proportion of the first double-stranded DNA therein.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.